Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

The deoxyhexose sugar fucose has an important fine-tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP-fucose, which is generated from GDP-mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP-mannose dehydratase (GMD; EC 4.2.1.47) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd-1 and gmd-2) and one GER-encoding gene (ger-1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.     

Recent progress in synthesis of carbohydrates with sugar nucleotide-dependent glycosyltransferases

Sugar nucleotide-dependent glycosyltransferases (GTs) are key enzymes that catalyze the formation of glycosidic bonds in nature. They have been increasingly applied in the synthesis of complex carbohydrates and glycoconjugates with or without in situ generation of sugar nucleotides. Human GTs are becoming more accessible and new bacterial GTs have been identified and characterized. An increasing number of crystal structures elucidated for GTs from mammalian and bacterial sources facilitate structure-based design of mutants as improved catalysts for synthesis. Automated platforms have also been developed for chemoenzymatic synthesis of carbohydrates. Recent progress in applying sugar nucleotide-dependent GTs in enzymatic and chemoenzymatic synthesis of mammalian glycans and glycoconjugates, bacterial surface glycans, and glycosylated natural products from bacteria and plants are reviewed.     

Sialic acid metabolism and sialyltransferases: natural functions and applications

Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.     

The role of eucaryotic cell surface glycoconjugates in DNA replication

Multiple forms of ricin have been isolated from castor bean seeds. Two forms, ricin-1 and ricin-2, differ in their isoelectric pI values and toxicity towards IMR-32 cells. Inhibition of IMR-32 DNA polymerase α₂ is more pronounced with ricin-1 (65%) than with ricin-2 (10%). Ricin B chain (pI = 5.2) isolated from ricin-1 binds to IMR-32 cell surfaces as well as inhibits DNA polymerase α₂ activity when studiedin vitro. The presence of galβ-linked glycoconjugates near the active site of IMR-32 DNA polymerase α₂ has been proposed. Replication modulators which bind to the glycose portion of the enzymes involved in the replication system may need a mandatory binding to cell surface glycoconjugates for their activity.     

Microbial beta-glucosidases: cloning,properties, and applications

Beta-glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.     

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.     

α2,6-Sialyltransferase Gene Transfection into a Human Glioma Cell Line (U373 MG) Results in Decreased Invasivity

Abstract: Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galβ1,4GlcNAc α2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated α3β1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125^fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.     

Fucosylation in prokaryotes and eukaryotes

Fucosylated carbohydrate structures are involved in a variety of biological and pathological processes in eukaryotic organisms including tissue development, angiogenesis, fertilization, cell adhesion, inflammation, and tumor metastasis. In contrast, fucosylation appears less common in prokaryotic organisms and has been suggested to be involved in molecular mimicry, adhesion, colonization, and modulating the host immune response. Fucosyltransferases (FucTs), present in both eukaryotic and prokaryotic organisms, are the enzymes responsible for the catalysis of fucose transfer from donor guanosine-diphosphate fucose to various acceptor molecules including oligosaccharides, glycoproteins, and glycolipids. To date, several subfamilies of mammalian FucTs have been well characterized; these enzymes are therefore delineated and used as models. Non-mammalian FucTs that possess different domain construction or display distinctive acceptor substrate specificity are highlighted. It is noteworthy that the glycoconjugates from plants and schistosomes contain some unusual fucose linkages, suggesting the presence of novel FucT subfamilies as yet to be characterized. Despite the very low sequence homology, striking functional similarity is exhibited between mammalian and Helicobacter pylori alpha1,3/4 FucTs, implying that these enzymes likely share a conserved mechanistic and structural basis for fucose transfer; such conserved functional features might also exist when comparing other FucT subfamilies from different origins. Fucosyltranferases are promising tools used in synthesis of fucosylated oligosaccharides and glycoconjugates, which show great potential in the treatment of infectious and inflammatory diseases and tumor metastasis.     

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.     

Microbial β-Glucosidases: Cloning,Properties, and Applications

ABSTRACT:?

β-Glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and amino-glucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial β-glucosidases, yeast β-glucosidases, thermostable fungal β-glucosidase, and the physiological functions, characteristics, and catalytic action of native β-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant β-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.     

Histochemistry of glycoconjugates in the secretory epithelium of the goat bulbourethral gland

To investigate the histochemical nature of the secretory epithelium lining the goat bulbourethral gland, glycoconjugates contained in these secretory epithelial cells have been studied by means of light- and electron-microscopic histochemistry. The methods employed involved a series of conventional staining and peroxidase-labeled lectin diaminobenzidine procedures together with combined selective methods such as digestion with enzymes. According to the results obtained in the present work, the secretory cells of the goat bulbourethral gland can be grouped into two types: cells with glycoconjugate-rich granules and those with less amounts of glycoconjugates. The gland is at least dual in the nature of its secretory activities.     

Molecular Evolution of Vertebrate Goose-Type Lysozyme Genes

We have found that mammalian genomes contain two lysozyme g genes. To better understand the function of the lysozyme g genes we have examined the evolution of this small gene family. The lysozyme g gene structure has been largely conserved during vertebrate evolution, except at the 5' end of the gene, which varies in number of exons. The expression pattern of the lysozyme g gene varies between species. The fish lysozyme g sequences, unlike bird and mammalian lysozyme g sequences, do not predict a signal peptide, suggesting that the encoded proteins are not secreted. The fish sequences also do not conserve cysteine residues that generate disulfide bridges in the secreted bird enzymes, supporting the hypothesis that the fish enzymes have an intracellular function. The signal peptide found in bird and mammalian lysozyme g genes may have been acquired as an exon in the ancestor of birds and mammals, or, alternatively, an exon encoding the signal peptide has been lost in fish. Both explanations account for the change in gene structure between fish and tetrapods. The mammalian lysozyme g sequences were found to have evolved at an accelerated rate, and to have not perfectly conserved the known active site catalytic triad of the bird enzymes. This observation suggests that the mammalian enzymes may have altered their biological function, as well.     

New approaches to enzymatic glycoside synthesis through directed evolution

The expanding field of glycobiology requires tools for the synthesis of structurally defined oligosaccharides and glycoconjugates, while any potential therapeutic applications of sugar-based derivates would require access to substantial quantities of such compounds. Classical chemical approaches are not well suited for such large-scale syntheses, thus enzymatic approaches are sought. Traditional routes to the enzymatic assembly of oligosaccharides have involved the use of either Nature’s own biosynthetic enzymes, the glycosyl transferases, or glycosidases run in transglycosylation mode. However, each approach has drawbacks that have limited its application. Glycosynthases are mutant glycosidases in which the catalytic nucleophile has been replaced by mutation, inactivating them as hydrolases. When used in conjunction with glycosyl fluorides of the opposite anomeric configuration to that of the substrate, these enzymes function as highly efficient transferases, frequently giving stoichiometric yields of products. Further improvements can be obtained through directed evolution of the gene encoding the enzyme in question, but this requires the ability to screen very large libraries of catalysts. In this review we survey new screening methods for the formation of glycosidic linkages using high-throughput techniques, such as FACS, chemical complementation, and robot-assisted ELISA assays. Enzymes were evolved to have higher catalytic activity with their natural substrates, to show altered substrate specificities or to be promiscuous for efficient application in oligosaccharide, glycolipid, and glycoprotein synthesis.     

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.     

Metabolic oligosaccharide engineering: perspectives, applications, and future directions

Many adhesion and signaling molecules critical for development, as well as surface markers implicated in diseases ranging from cancer to influenza, contain oligosaccharides that modify their functions. Inside a cell, complex glycosylation pathways assemble these oligosaccharides and attach them to proteins and lipids as they traffic to the cell surface. Until recently, practical technologies to manipulate glycosylation have lagged unlike the molecular biologic and genetic methods available to intervene in nucleic acid and protein biochemistry; now, metabolic oligosaccharide engineering shows promise for manipulating glycosylation. In this methodology, exogenously-supplied non-natural sugars intercept biosynthetic pathways and exploit the remarkable ability of many of the enzymes involved in glycosylation to process metabolites with slightly altered chemical structures. To date, non-natural forms of sialic acid, GalNAc, GlcNAc, and fucose have been incorporated into glycoconjugates that appear on the cell surface; in addition O-GlcNAc protein modification involved in intracellular signaling has been tagged with modified forms of this sugar. Reactive functional groups, including ketones, azides, and thiols, have been incorporated into glycoconjugates and thereby provide chemical 'tags' that can be used for diverse purposes ranging from drug delivery to new modes of carbohydrate-based cell adhesion that can be used to control stem cell destiny. Finally, strategies for further engineering non-natural sugars to improve their pharmacological properties and provide complementary biological activities, such as addition of short chain fatty acids, are discussed in this article.     

Polymeric glycoconjugates protect and activate macrophages to promote killing of <Emphasis Type="Italic">Bacillus cereus</Emphasis> spores during phagocytosis

Diseases caused by Bacillus spores might be attenuated if macrophages were able to kill the spores on exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for modulation of phagocytosis of B. cereus spores. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores during phagocytosis. In the presence of glycoconjugates, however, the macrophages efficiently killed the organisms. The glycoconjugates were shown to have a protective influence, sparing macrophages from spore-induced cell death. Very low concentrations of the glycoconjugates prevented macrophage cell death, as shown by lactate dehydrogenase (LDH) release and trypan blue assays. Increased levels of inducible nitric oxide (NO) production by the macrophages in the presence of glycoconjugates suggested that the glycoconjugates provide an activation signal to the macrophages. These results suggest that glycoconjugates promote the killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level. Polymeric glycoconjugates may suggest novel approaches to improve existing vaccines as well as prevent and treat infections incurred through either B. cereus or B. anthracis spores.     

Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme

Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E. coli. We have used synthetic acceptors to probe the acceptor requirement of these enzymes and to examine the basic enzymology. The minimum number of sialic acid residues (Neu5Ac) on the acceptor for activity in vitro was shown to be 2 for both enzymes, but a large increase in activity was seen if the acceptor had three Neu5Ac residues. The polysialyltransferase from N. meningitidis generated longer reaction products than the enzyme from E. coli on FCHASE acceptors. Examination of the products showed them to be a heterogeneous mixture, but products with >50 Neu5Ac residues could be seen using capillary zone electrophoresis analyses. In addition we made fusion proteins of these polysialyltransferase enzymes with the bifunctional alpha-2,3/alpha-2,8-sialyltransferase from Campylobacter jejuni to create self priming polysialyltransferases. These bifunctional sialyltransferases utilized various synthetic disaccharide acceptors with a terminal galactose, and we demonstrate here that the PST enzyme from N. meningitidis and its fusion protein with the C. jejuni sialyltransferase can be used to create polysialic acid on O-linked glycopeptides.     

New insights into heparan sulphate biosynthesis from the study of mutant mice

Heparan sulphate (HS) is an essential co-receptor for a number of growth factors, morphogens and adhesion proteins. The biosynthetic modifications involved in the generation of a mature HS chain may determine the strength and outcome of HS-ligand interactions. These modifications are catalysed by a complex family of enzymes, some of which occur as multiple gene products. Various mutant mice have now been generated, which lack the function of isolated components of the HS biosynthetic pathway. In this discussion, we outline the key findings of these studies, and use them to put into context our own work concerning the structure of the HS generated by the Hs2st -/- mice.     

Comparative aspects of glycosyltransferases

Glycosyltransferases, the enzymes that build oligosaccharides and glycoconjugates, have received much interest in recent years owing to their biological functions and their potential uses in biotechnology. Despite the fact that many glycosyltransferases recognize similar donor or acceptor substrates, there is surprisingly limited sequence identity between different classes. On the one hand, the glycosyltransferases are found in a large number of families, by sequence-based classification. On the other hand, only two structural folds have been identified among the fewer than one dozen glycosyltransferases that have been crystallized at present. Detection of conserved motifs that have a direct role in the functional aspects of glycosyltransferases is one approach for identifying remote similarity. With the availability of more crystal structures, the use of the fold-recognition approach is also very promising.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.